During the highly conserved process of cell division, chromosomes must first capture spindle microtubules. The organelle responsible for this capture is the kinetochore, which is assembled at a distinct chromosomal site known as the centromere. Despite tremendous progress in understanding the segregation mechanisms in budding yeast, which involves small kinetochores, relatively little is known about the more complex mammalian kinetochore. Disturbances in chromosome segregation probably underlie many pathological conditions, including spontaneous abortions, trisomy, and tumor progression. Thus understanding chromosome segregation is of profound importance for human health. This proposal addresses the mechanism of kinetochore function, which is a primary structural problem in mitosis. Following the successful approaches used in yeast, we will identify what part of the human centromere is responsible for assembling a kinetochore. Cytogenetic evidence for functional redundancy of higher centromeres suggests multiple subdomains exist within this DNA. We have proposed experiments to determine whether this functional redundancy is a consequence of independent DNA domains equally capable of assembling kinetochore proteins. The second major goal of this proposal is to clone novel kinetochore proteins associated with the interphase centromere- kinetochore complex from mammalian cells and to determine their role in kinetochore assembly. These questions will be addressed using cell-free Xenopus embryonic extracts to assemble kinetochore proteins onto defined regions of the human Y centromere. Human kinetochore proteins purified from interphase kinetochore complexes will be cloned and incorporated into the assembly system. The ability of CENP-C, a human kinetochore protein, to assemble into Xenopus kinetochores suggests the feasibility of this approach. This cell-free system will present unique Opportunities to address the function of specific kinetochore proteins through addition/depletion experiments, to systematically define DNA sequences involved in kinetochore assembly, and to study the regulation of these processes during the cell cycle.